Antibiotic derivatives of gentamicin c2

ABSTRACT

DERIVATIVES OF GENTAMICIN C2 HAVE BEEN PREPARED WHICH POSSES SUBSTANTIALLY IMPROVED ANTIBACTERIAL ACTIVITY. AN EXAMPLE OF SUCH AN AGENT IS 1-(L-(-)-Y-AMINO-A-HYDROXYBUTYRYL)-GENTAMICIN C2 (III, BB-K75).

"United States Patent 3,796,698 ANTIBIOTIC DERIVATIVES 0F GENTAMICIN C Takayuki Naito, Susumu Nakagawa, and Yoshio Abe, Tokyo, Japan, assignors to Bristol-Myers Company, New York, N.Y. No Drawing. Filed Oct. 18, 1972, Ser. No. 298,748 Int. Cl. C07c 129/18 US. Cl. 260-210 AB 8 Claims ABSTRACT OF THE DISCLOSURE Derivatives of gentamicin C have been prepared which possess substantially improved antibacterial activity. An example of such an agent is l-[L-()-'y-amino-vt-hydroxybutyryl]-gentamicin C [HI, BB-K75].

BACKGROUND OF THE'INVENTION (1) Field of the invention This invention relates to a semisynthetic l-substituted derivative of gentamicin C said compound being prepared by acylating the l-amino-function of gentamicin C, with a 'y-amino-a-hydroxybutyryl moiety.

(2) Description of the prior art (A) Gentamicin (gentamycin) and the fermentation thereof is the subject matter of US. Pat. Nos. 3,091,572 which issued May 28, 1962 and 3,136,704 which issued June 9, 1964. The patents describe the fermentation of gentamicin complexes from Micromonospora purpurea, NNRL 2953 and Micromonospora echinospora, NNRL 2985. Variants thereof have also been deposited in the stock culture collection of the United States Department of Agriculture, Northern Utilization Research and Development Division, Peoria, Ill. as M. echinospora var. ferruginea, NNRL 2995 and M. echinospora var. pallida, NNRL 2996.

(B) Gentamicin is further described in the Merck Index, 8th edition, p. 485. The two major components, gentamicin C and C are described therein.

SUMMARY OF THE INVENTION The compound having the formula in which R is L-()-'y-amino-u-hydroxybutyryl; or a nontoxic pharmaceutically acceptable acid addition salt thereof is a valuable antibacterial agent.

3,796,698 Patented Mar. 12, 1974 "Ice This invention relates to semi-synthetic derivatives of gentamicin C said compound being known as 1-[L-()- 'y-amino-a-hydroxybutyryl]-gentamicin C and having the formula in which R is L-( --)-'y-amino-a-hydroxybutyryl; or a non toxic pharmaceutically acceptable acid addition salt thereof.

For the purpose of this disclosure, the term nontoxic pharmaceutically acceptable acid addition salt shall mean a mono, di-, tri-, tetra, or penta salt formed by the interaction of 1 molecule of Compound III with 1-5 moles of a nontoxic, pharmaceutically acceptable acid. Included among these acids are acetic, hydrochloric, sulfuric, maleic, phosphoric, nitric, hydrobromic, ascorbic, malic and citric acid, and those other acids commonly used to make salts of amine containing pharmaceuticals.

Gentamicin C is fermented as a complex of three closely related components designated C C and C For the purpose of this application we are only concerned with gentamicin C the starting material of the compounds of the instant invention, which has the formula The compounds of the present invention are prepared by the following diagrammatic scheme:

N-hydroxysucoinimide ester of L-(-)- 'y-benzyloxycarbonylamino-u-hydroxybutyric acid (A) Gentamicin C.

compound II 3 lPd/c CH CPI-NR III A preferred embodiment of the present invention is the comuound having the formula in which R is L-()-'y-amino-a-hydroxybutyryl or L-(-)-'y-benzyloxycarbonylamino-a-hydroxybutyryl; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

A more preferred embodiment is the compound of Formula IV in which R is L-(-)-'y-benzyloxycarbonylamino-a-hydroxybutyryl; or an acid addition salt thereof.

A most preferred embodiment is the compound of Formula IV in which R is L-(-)-'y-amino-u-hydroxybutyryl; or a nontoxic pharmaceutically acceptable salt thereof. 1

The most preferred embodiment is the compound of Formula IV in which R is L-()-'y-amino--hydroxybutyryl; or the mono or disulfate salt thereof.

Other most preferred embodiments are the hydro chloride, acetate, maleate, citrate, ascorbate, nitrate or phosphate salts of Compound IV.

The objectives of the present invention have been achieved, by the provision according to the present invention of the process for the preparation of the compound having the formula III in which R is L-()-'y-amino-a-hydroxybutyryl; or a nontoxic pharmaceutically acceptable acid addition salt thereof; which process comprises the consecutive steps of (A) Acylating gentamicin C With an acylating agent having the formula OH WNHCH1CHQ-(iJH B-M VII in which W is a radical selected from the group comprising Rt OH;

but preferably in which R and R are alike or different and each is H, F, Cl, Br, N0 OH, (lower)alkyl or ((lower)alkoxy, X is chloro, bromo or iodo and M is a radical selected from the group comprising O N O l N O, l but preferably 0-N N like but preferably 1:1 water-ethylene glycol dimethyl ether, to produce a compound of the formula in which W is as above; and

(B) Removing the blocking group W from the Com- 7 pound II by methods commonly known in the art, and

preferably when W is a radical of the formula by hydrogenating Compound II with hydrogen in the presence of a metal catalyst, preferably selected from the group comprising palladium, platinum, Raney nickel, rhodium, ruthenium and nickel, but preferably palladium, and most preferably palladium on charcoal, in a waterwater miscible solvent system, preferably selected from the group comprising water and dioxane, tetrahydrofuran, ethyleneglycol dimethyl ether, propyleneglycol dirnethyl ether, or the like, but preferably 1:1 wateniioxane, and preferably in the presence of a catalytic amount of glacial acetic acid to produce the compound of Formula III.

It should be apparent to those knowledgable in the art that other agents can be used in the process above to acylate the amine functions of the intermediate compounds of the instant invention. This disclosure is meant to include all such acylating agents that produce labile amine blocking groups, said labile blocking groups commonly employed in the synthesis of peptides. The labile blocking groups must be readily removable by methods commonly known in the art. Examples of said labile blocking groups and their removal can be found in the review of A. Kapoor, I. Pharm.Sciences 59, pp. 1-27 (1970). Functional equivalents as acylating agent for primary amine groups would include corresponding carboxylic chlorides, bromides, acid anhydrides, including mixed anhydrides and particularly the mixed anhydrides prepared from stronger acids such as the lower aliphatic monoesters of carbonic acid, of alkyl and aryl sulfonic acids and of more hindered acids such as diphenylacetic acid. In addition, an acid azide or an active ester of thioester (e.g., with p-nitrophenol, 2,4-dinitrophenol, thiophenol, thioacetic acid) may be used or the free acid itself may be coupled with the gentamicin C derivative after first reacting said free acid with N,N'-dimethylchloroforminium chloride [cf. Great Britain 1,008,170 and Novak and Weichet, Experientia XXI/6, 360 (1965)] or of alkynylamine reagent [cf. R. Buijile and H. G. Viehe, Angew. Chem., International Edition 3, 582 (1964)], or of a ketenimine reagent [cf. C. L. Stevens and M. E. Munk, J. Amer. Chem. Soc. 80, 4065 (1958)] or of an isoxazolium salt regent [cf. R. B. Woodward, R. A. Olofson and H. Mayer, J. Amer. Chem. Soc., 83, 1010 (1961)]. Another equivalent of the acid chloride is a corresponding azolide, i.e., an amide of the corresponding acid whose amide nitrogen is a member of a quasiaromatic five membered ring containing at least two nitrogen atoms, i.e., imidazole, pyrazole, the triazoles, benzimidazole, benzotriazole and their substituted derivatives. As an example of the general method for the preparation of an azolide, N,N-car-bonyldiimidazole is reacted with a carboxylic acid in equimolar proportions at room temperature in tetrahydrofuran, chloroform, dimethylformamide or a similar inert solvent to form the carboxylic acid imidazolide in practically quantitative yield with liberation of carbon dioxide and one mole of imidazole. Dicarboxylic acids yield diimidazolides. The by-product, imidazole, precipitates and may be separated and the imidazolide isolated, but this is not essential. These reactions are well-known in the art [cf. US. Pat. Nos. 3,079,314; 3,117,126 and 3,129,224 and British Pat. Nos. 932,644; 957,570 and 959,054].

Compound III, 1-[L-()-'y-amino-a-hydroxybutyryl]- genta-micin C possesses excellent antibacterial activity. Illustrated below is a table showing the minimal inhibitory concentrations (MICs) of gentamicin C Compound III (BB-K), and two other related monoand diacylated derivatives of gentamicin C against a variety of Grampositive and Gram-negative bacteria as determined by the two-fold agar dilution method.

TABLE 1 In vitro antimicrobial activity of gentamic in C derivatives Genta- Bristol miein Strain No. BB-K74 BB-K75 BB-K76 G:

E. coli NIHJ 3. 1 0. 8 6. 3 0. 8 E. coli Juhl- A15119 3.1 0.8 6. 3 0.8 E. coli JuhL. A15169 3. 1 0. 8 6. 3 0. 8 E. coli .Tuhl KM-R l A20363 3.1 0. 4 6.3 0. 8 E. coli Juhl-.. A9844 3. 1 0. 4 6. 3 0. 8 E. coli .l'uhl KM-R l A20365 0.8 0.1 0.8 0.1 E. coli K-12 1. 6 0. 2 3. 1 0. 4 E. colt K-12 KM-R 0.8 0.2 3. 1 0.4 E. coli K-12 -KM-R l A20665 1. 6 0. 2 3. 1 0. 4 E. coli W677 1820684: 0. 8 0. 2 6.3 0. 8 E. coli SIR/W677 A20683 12. 5 0. 4 3.1 12.5 K. pneumoniae D11 0. 8 0. l 0. 8 0.2 r ype 22 A20680 12.5 0. 8 6.3 25 SET. marflsrme A20019 3. 1 1. 6 6. 3 0. 8 Fe. m D-l) 25 100 6. 3 P8. ueruginosa H9 D-113 KM-R 100 100 100 12. 5 Ps. aemginosa H9 A9923 100 100 100 12. 5 Ps. aeruginosa H9 A9930 3. 1 0. 8 6.3 0. 4 .Ps. aerugz'nosa H9 A15150 100 100 100 25 Pa. uw H9 A15194 100 50 100 12.5 P8. aeruginosa H9 GM-R A20717 100 100 100 100 Pa. aerugz'noaa H9 GM-R 9 20718 00 100 100 100 Pa. aeruginoaa H6 D-114 NM-R 100 25 100 6.3

TABLE 1-Con.tlnued Genta- Bristol micin Strain No. BIB-K74 BBK75 BB-K76 Oz Pr. vulgaria.. A9436 1. 6 0. 8 3. 1 0. 4 Pr. vulgar A9526 3. 1 0.8 6.3 0. 4 Pr. mirabilz's- A9554 12. 5 1. e 25 o. s Pr. mirabilis- A9900 6. 3 1. e 25 o. s Pr. morgrmii A9553 3. 1 1. 6 25 0. 8 Pr. morgvmii. A20031 6. 3 3. 1 25 0. 8 S. aureus Smi A15167 0. 8 0. 2 3. 1 0. 2 S. aureus 209P SM-R 1 12. 5 3. 1 25 0.8 S. aureus KM-R A20239 3. 1 0.8 6.3 0. 4

1 KMR is kanamycin resistant. 1 SM-R is streptomycin resistant. l GM-R is gentamicin resistant. 4 NM-R is neomycin resistant.

Since gentamicin C has several amino functions capa- Compound III is valuable as an antibacterial agent, ble of acylation when treated with the N-hydroxysuccinnutritional supplement in animal feeds, therapeutic agent imide ester of L-()-' -benzyloxycarbonylaminoa -hyin poultry and animals, including man, and is especially droxybutyric acid, varying quantities of difierent prodvaluable in the treatment of infectious diseases caused nets are obtained depending upon the site of acylation by Gram-positive and Gram-negative bacterial. when treated according to the following scheme: Compound III is useful as an adjunctive treatment for Gentamkin a preoperative sterilization of the bowel when administered 2 orally. Both aerobic and anaerobic flora which are susceptible to this drug are reduced in the large intestine. cbz-micir ca cifcoom I When accompanied by adequate mechanical cleansing, it

$ 0 is useful in preparing for colonic surgery.

Compound III is elfective in the treatment of systemic l bacterial infections when administered parenterally in the dosage range of about 250 mg. to about 3000 mg. per dayl CG 0 CO1 in divided doses three or four times a day. Generally the fig ggg compound is effective when administered at a dosage of H about 5.0 to 7.5 mg./kg. of body weight every 12 hours. i 3 Genamicin C is a complex of at least three major com- 2 ponents difiering only the R substituent found on the 5 position of the dideoxy-sugar moiety of the gentamicin 33447 $5475 nucleus as shown below:

H on NIHJOC'HCH CiH 40 c H5 or: us 8 01i BIB-K75 It can be seen that the process produced three different HABA-substituted gentamicin C derivatives, herein designated BBK74, BB-K75 and BBK76. BB-K75 was determined to be 1-[L-(-)-'y-amino-a-hydroxybutyryl]-gentamicin C (III).

BBK75, the most active product, is equally active or a more active than gentamicin C against E. coli and K. R pneumonz'ae strains tested so far. It is 16-32 times more Gentamicin C -OH-NH-CH; active against E. coli A20683 and K. pneumoniae A20680 (3H8 which are known to inactivate gentamicin C by 2-ade- O nylation, but 2-8 times less active against strains of Gentamicin Pseudomonas, Proteus and Staphylococcus. CH:

BB-K74 is a monoacylated gentamicin C derivative Gentamicin on and BB-K76 a dracylated gentamicin C derivatlve. The Gentami in C bt d f th 1 b acylation sites of these two compounds have not been f 2 was 0 mm mm comp ex y determined. the ollowmg procedure.

BBK74 and BB-K76 were also similar in their anti- SEPARATION OF GENTAMIClIN C COMPLEX bactenal feamr es to t of B3405 i 9 less F' Gentamicin C complex (4.10 g.) as the free base was tlve. In comparison with the parent antibiotic gentamlcm fr actionated using a preparative counter current distri- C all of the acylation products lsolated here showed l improved activity a ainst the two gentamicin-resistant butlon (C0D) apparatus (100 mlxso tubes) wlth solvent system of CHCl --MeOH17% aqueous NH OH strains (E. coli A20683 and K. penumoniae A20680), whereas the activity decreased to almost nothing against the gentamicin-resistant strains of Pseudomonas.

BBK74, BB-K and BB-K76 were confirmed by TLC (thin layer chromatography) to liberate gentamicin C and L-I-IABA on hydrolysis with 0.5 N aqueous NaOH solution at C. for one hour. Thus these three compounds were proved to be gentamicin C derivatives acylated with L-HABA. This fact, as well as the presence of an amide-carbonyl band in the -IR (infrared) spectra, show that all of them are gentamicin C derivatives.

(2:1:1) to give the fractions shown below.

Amount Identifirecovcation cred, g. (TLC 1.46 Crmain. 0.72 0.02.

1.20 C2+Clm 0.87 0;,main.

1 Cellulose thin-layer plate solvent system: lower layer of 011013-- Me2%)IEI17% NH4OH (2:1:1): Rt: gentamicin 0 (0.69), C: (0.39), 0;,

Fraction Fractions I and H were combined and distributed again by the same CCD system as above to give 1.94 g. of the gentamicin C component which was further purified by a Dowex 1 X2 column chromatography yielding 1.70 g. of substantially pure gentamicin C Other fractions were purified similarly by CCD and Dowex 1 X2 chromatography to give 1.13 g. of gentamicin C and 0.62 g. of gentamicin C Dowex 1 X2 is a resin comprised of a polystyrene backbone to which is attached benzyltrimethylammonium chloride and 2% of added divinylbenzene as a crosslinking agent during polymerization of the polystyrene. It is supplied as beads.

EXAMPLES Example 1.Preparation of L-()-' -benzyloxycarbonylamino-a-hydroxybutyric acid (VI) L 'y amino u hydroxybutyric acid (7.4 g., 0.062 mole) was added to a solution of 5.2 g. (0.13 mole) of sodium hydroxide in 50 ml. of water. To the stirred solution was added dropwise at -5 C. over a period of 0.5 hour, 11.7 g. (0.068 mole) of carbobenzoxy chloride and the mixture aws continued to stir for one hour at the same temperature. The reaction mixture was washed with 50 ml. of ether, adjusted to pH 2 with dilute hydrochloric acid and extracted with four 80-m1. portions of ether. The ethereal extracts were combined, washed with a small amount of saturated sodium chloride solution, dried with anhydrous sodium sulfate and filtered. The filtrate was evaporated in vacuo and the resulting residue was crystallized from benzene to give 11.6 g. (74%) of colorless plates; melting point 78.5- 79.5 C., [a] =4.5 (c. =2, CH OH). Infrared (IR) [KBr]: IR (KBr) C=O 1740, 1690 cm. Nuclear Magnetic Resonance (NMR) (acetone-d m (in p.p.m. from TMS) 2.0 (2H, m.), 3.29 (2H, d-d, 1:67 and 12 Hz.), 4.16 (1H, dd, 1:45 and 8 Hz), 4.99 (2H, s.), 6.2 (2H, broad), 7.21 (H, s.).

Analysis.-Calcd for C H NO (percent): C, 56.91; H, 5.97; N, 5.53. Found (percent): C, 56.66; H, 5.97; N, 5.47.

Example 2.N-hydroxysuccinimide ester of L-()-'ybenzyloxycarbonylamino-u-hydroxybutyric acid (VII) A solution of 10.6 g. (0.042 mole) of VI and 4.8 g. (0.042 mole) of N-hydroxysuccinimide in 200 ml. of ethyl acetate was cooled to 0 C. and then 8.6 g. (0.042 mole) of dicyclohexylcarbodiimide was added. The mixture was kept overnight in a refrigerator. The dicyclohexylurea which separated was filtered 011 and the filtrate was concentrated to about 50 ml. under reduced pressure to give colorless crystals of VII which were collected by filtration; 6.4 g., M.P. 121-1225" C. The filtrate was evaporated to dryness in vacuo and the crystalline residue was washed with 20 m1. of a benzenen-hcxane-mixturc to give an additional amount of VII. The total yield was 13.4 g. (92%). [a] |-1.5 (c. =2, CHCl IR (KBr) 'yC=O 1810, 1755, 1740, 1680 cmf NMR (acetone-d w (in p.p.m. from TMS) 2.0, (2H,

' m.), 2.83 (2H, s.), 3.37 (2H, d-d, 1:65 and 12.5 HZ.),

4.56 (1H, m.), 4.99 (2H, s.), 6.3 (2H, broad), 7.23 (5H, s.).

Analysis.Calcd for C H N O (percent): C, 54.85; H, 5.18; N, 8.00. Found (percent): C, 54.79, 54.70; H, 5.21, 5.20; N, 8.14, 8.12.

Example 3.Preparation of 1-[L-(-)-'y-aminou-hydroxybutyryl]-gentamicin C (HI) To a stirred solution of 325 mg. (0.7 mmole) of gentamicin C in 40 ml. of 75% aqueous THF (tetrahydrofuran) was added 245 mg. (0.7 mmole) of N-hydroxysuccinimide ester of L-()-'y-benzyloxycarbonylamino-u-hy- G. W. Anderson et 8.1., J. Am. Chem. Soc, 86, 1839 (1964).

droxybutyric acid. The mixture was stirred overnight at room temperature and hydrogenated with 10% palladium on charcoal for seven hours at ordinary temperature and pressure. The reaction mixture was filtered, the filtrate was evaporated in vacuo to remove the organic solvent and the resulting concentrate charged on a column of Amberlite CG-50 ion-exchanger (NH form, 10 ml.), which was washed with 60 ml. of water and eluted successively with 0.1 N NH4OH (480 ml.), 0.2 N NH OH (450 ml.) add 0.5 N NH4OH (800 ml.). The eluate was collected in 10-ml. fractions. The fractions were monitored by ninhydrin spot test, disc assay and TLC, and grouped as follows. Each group was concentrated under reduced pressure and lyophilized.

Weight isolated, mg. Compound 28 Crude BB-K73. A mixture of BB-K75. 18 BB-K76.

Fraction number Eluted with- 37-65 0.1-0.2 N NHAOH 101-130 0.5 N NHlOH Group 3 161-170 0.5 N NH4OH Properties 70 O M.P., C. Code No (dee.) Bi 1 cm.-

TLC: silica gel plate, CHOla-MeOH-28% NH OH-H20 (2:8:2z1).

Amberlite CG 50 is the trade name for the chromatographic grade of a weakly acidic cationic exchange resin of a carboxylic-polymethacrylic type.

Example 4.--Preparation of L-(-)-'y-amino-a-hydroxybutyric acid from ambutyrosin A or B or mixtures thereof Ambutyrosin A (5.0 gm.) [U.S. Pat. No. 3,541,078, issued Nov. 17, 1970] was refluxed with 160 ml. of 0.5 N sodium hydroxide for one hour. The hydrolysate was neutralized with 6 N HCl and chromatographed on a column of CG-50 (NH type). The desired L-(-)- -amino-a-hydroxybutyric acid was isolated by developing the column with water and removing the water by freeze drying. The L-()'y-amino-a-hydroxybutyric acid is characterized as a crystalline material having M.P. of 212.5-214.5 C. [column 2, lines 31-38, U.S. Pat. No. 3,541,078].

Example 5.Preparation of L-(-)-'y-'amino-a-hydroxybutyric acid from DL-u-hydroxy- -phthalirnodbutyric acid (A) Dehydroabietylammom'um L-a-hydroxy-v-phthalimidobutyrate: To a solution of 25 g. (0.1 mole) of 2-hydroxy- -phthalimidobutyric acid in 200 ml. of ethanol 'was added a solution of 29 g. (0.1 mole) of dehydroabietylamine in 130 ml. of ethanol. The solution was shaken vigorously for a minute and stood at room temperature for five hours during which time fine needles crystallized out. The crystals were collected by filtration, washed with 50 ml. of ethanol and air dried to obtain 30.1 g. (56%) of a diastereomer of the dehydroabietylamine salt. M.P. 93-94 C. [u] +15 (C. 2.5, MeOH).

Recrystallization from 300 ml. of ethanol gave 23.2 g.

(43%) of the pure product. M.P. 94-95 C. [111 +10.8 (C. 2.5, MeOH). Further recrystallization did not change the melting point and the specific rotation.

Y. Saito et al., Tetrahedron Letters, 1970, 4863.

Analysis.-Oalcd for C H N O -H O (percent): C, 69.54; H, 8.02; N, 5.07. Found (percent): C, 69.58; H, 8.08; N, 5.07.

(B) L-(--)-'y-amino-o -hydroxybutyric acid: To a solution of 1.5 g. (0.014 mole) of sodium carbonate in 40 m1. of water were added 5.3 g. (0.01 mole) of dehydroabietylammonium-L-u-hydroxy-y-phthalimidobutyrate and 60 ml. of ether. The mixture was shaken vigorously until all of the solid had dissolved. The ether layer was separated. The aqueous solution was washed twice with 20-ml. portions of ether and evaporated to 15 ml. under reduced pressure. To the concentrate was added ml. of concentrated hydrochloric acid and the mixture was refluxed for ten hours. After cooling, separated phthalic acid was removed by filtration. The filtrate was evaporated under reduced pressure. The residue was dissolved in 10 ml. of water and the solution was evaporated to dryness. This operation was repeated twice to remove excess hydrochloric acid. The residual syrup was dissolved in 10 ml. of water and filtered to remove a small amount of insoluble phthalic acid. The filtrate was adsorbed on a column of IR-20 (H'*', 1 cm. x 35 cm.), the column was washed with 300 ml. of water and eluted with 1 N ammonium hydroxide solution. The eluate was collected in l5-ml. fraction. The ninhydrin positive fractions 10 to 16 were combined and evaporated under reduced pressure to give a syrup which crystallized gradually. The crystals were triturated with ethanol, filtered and dried in a vacuum desiccator to give 0.78 g. (66%) of L-()-'y-aminoa-hydroxybutyric acid, M.P. 206-207 C. [11],; 29 (C. 2.5, H 0). The IR spectrum was identical with the authentic sample which was obtained from ambutyrosin.

Amberlite IR-120 is the trade name for a high density nuclear sulfonic acid type cationic exchange resin supplied in either hydrogen or sodium form as beads-l6-50 mesh.

Example 6.-Preparation of the monosulfate salt of l-[L- q-amino-u-hydroxybutyryl] gentamicin C One mole of 1-[L-(--)-'y-amino-ahydroxybutyryl]- gentamicin C is dissolved in 1 to 3 liters of Water. The solution is filtered to remove any undissolved solids. To the chilled and stirred solution is added one mole of sulfuric acid dissolved in 500 ml. of water. The mixture is allowed to stir for 30 minutes, following which cold ethanol is added to the mixture till precipitation occurs. The solids are collected by filtration and are determined to be the desired monosulfate.

Example 7.Preparation of the disulfate salt of l-[L- y-amino-a-hydmxybutyryl] gentamicin C;

One mole of 1-[L-(-)-y-amino-a-hydroxybutyryl]- gentamicin C is dissolved in 1 to 3 liters of water. The

CH CH-NH on at a in which R is L-(-)-y-amino-a-hydroxybutyryl or L- )-'y-benzyloxycarbonylamino-a-hydroxybutyryl; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

2. The compound of claim 1 wherein R is L-(-)-'ybenzyloxycarbonylamino-u-hydroxybutyryl; or an acid addition salt thereof.

3. The compound of claim 1 wherein R is L-(-)-'yamino-a-hydroxybutyryl; or a nontoxic pharmaceutically acceptable acid addition salt thereof.

4. The monosulfate salt of the compound of claim 3.

5. The disulfate salt of the compound of claim' 3.

6. The mono or polyhydrates of the compound of claim 3.

7. The mono or polyhydrates of the compounds of claim 4.

8. The mono or polyhydrates of the compounds of claim 5.

References Cited UNITED STATES PATENTS 3,268,508 8/ 1966 Sugazawa et al. 260210 K 3,647,779 3/1972 Schmitz 260-210 AB 3,651,042 3/1972. Marquez et a1. 260210 AB JOHNNIE R. BROWN, Primary Examiner US. Cl. X.R. 

